Week 11 — Bio Production
Bio Production
Cell-Free Systems, Fluorescent Proteins & Master Mix Design
Cell-Free Reagent Breakdown
The broken-down remains of E. coli cells — containing DNA, RNA, and critically the protein machinery like T7 RNA Polymerase that performs the transcription and translation of our desired protein.
Provides the building blocks for RNA and DNA synthesis during transcription, drawing on ATP/GTP derived from guanine and glucose. Also supplies the amino acids and energy needed for protein synthesis downstream.
The raw material for actually building the coded proteins. Notably, cysteine — with its –SH side chain — provides the reducing capability required for the correct functioning of the enzymes involved.
The electron carrier system required for NAD-dependent reactions: metabolic processes, oxidative reactions that generate NADH as a byproduct, and as the precursor for many essential enzymes.
Brings the solution to the required volume and dilution factor. The nuclease-free part matters — ordinary water can carry nuclease enzymes that will happily degrade the DNA in the reaction.
The key difference is how each system supplies and sustains ATP for transcription and translation. PEP-based systems are fast and high-powered — but short-lived. ATP generation is rapid, which means rapid depletion of the energy source and a drifting pH as pyruvate accumulates.
Long-incubation NMP systems are slower by design, drawing on a broader nutrient mix that takes longer to metabolise into ATP. The payoff is a lower initial protein expression rate, but also lower phosphate accumulation — meaning better pH stability over the course of the reaction.
Guanosine Monophosphate (GMP) is simply a guanine molecule bound to a phosphate group. It can be dephosphorylated to release the guanine nucleotide — and this dephosphorylation step also releases useful energy, so it's doing double duty.
Cell-Free Master Mix Design
1 — Fluorescent Protein Properties
Engineered specifically to resist misfolding when fused to poorly-folded proteins. Folds in under 10 minutes and tolerates the chaperone-free, crowded environment of cell-free lysates far better than earlier GFP variants — its folding robustness is its defining trait.
A relatively slow-maturing monomer with low acid sensitivity. The notable issue: red FPs pass through green chromophore intermediates during maturation — a fraction of mRFP1 may never fully mature past this green state, or becomes trapped as a dead-end product entirely.
Complex multi-step chromophore maturation involving multiple oxidation steps — making it significantly slower overall. Fluorescence signal will substantially lag behind protein synthesis, making mKO2 a poor kinetic reporter, though perfectly fine for endpoint measurements.
Standout property: an exceptionally high quantum yield combined with acid stability. Its very low pKa of 3.1 means fluorescence is essentially independent of pH across a huge range — a robust reporter in variable-pH environments.
A single amino acid substitution — T74I — drives a marked maturation acceleration, making it one of the fastest-maturing red FPs available and an excellent kinetic reporter. The trade-off is a moderate reduction in fluorescence quantum yield.
A recently engineered blue FP derived from the Anthozoa protein eqFP611 via mRuby3. Its tendency toward aggregate formation confers resistance to acidity and proteases in mammalian cells — but in a cell-free context, without cell growth diluting things, aggregation could trap a meaningful proportion of translated Electra2 in non-fluorescent or insoluble fractions, reducing effective signal.
2 — Reagent Adjustment Hypothesis
Sodium percarbonate (Na₂CO₃·1.5H₂O₂) at 0.5–2 mM as a slow-release oxygen source.
mKO2 chromophore maturation requires two sequential oxidation steps — acylimine formation then third-ring cyclisation — giving it a ~135 minute maturation half-time. In a sealed cell-free reaction, oxygen is consumed both by this multi-step chemistry and by the energy regeneration system, creating a hypoxic microenvironment that stalls maturation and traps translated protein in a non-fluorescent intermediate state. Over 36 hours, this oxygen depletion is cumulative and severe.
Sustained O₂ availability will drive more translated mKO2 through both oxidation steps to the fully mature orange-emitting state, increasing plateau fluorescence at 36 hours. Because mKO2 requires two oxidation steps versus one for GFP-family proteins, it is disproportionately sensitive to O₂ limitation — making this a mechanistically specific intervention rather than a generic tweak. If the reaction is run in an open-well format where O₂ is not limiting, no effect should be observed, which would falsify the oxygen-depletion mechanism.
4 — Reaction Composition Per Well
| E. Coli Lysate | 6 µL |
| 2X Optimised Master Mix | 10 µL |
| FP DNA Template | 2 µL |
| Custom Reagent Supplements | 2 µL |
| Total | 20 µL |
Analysis of fluorescence data from the global experiment will follow approximately one week after data is returned. Date TBD.